吉西他滨耐药细胞系H460/Gem及其亲代细胞NCI-H460之间的不同

Objective: To discuss the difference between multi-drug resistant cell line H460/Gem and its parental cell NCI-H460 on the basis of establishment of human gemcitabine-resistant cell line H460/Gem so as to elaborate the possible mech-anisms of gemcitabine resistance. Methods: Human gemcitabine-resistant non-small cell lung cancer cell line H460/Gem was established by 2/3 clinical serous peak concentration gemcitabine intermittent selection from its parental cell human large cell lung carcinoma cell line NCI-H460 which was sensitive to gemcitabine. During the course of inducement, we had monitored their morphology, checked their resistance indexes and resistant pedigree by MTT method, gathered their growth curves and calculated their doubling time, examined their DNA contents and cell cycles by FCM; at the same time, we had measured its expressions of P53, EGFR, c-erb-B-2, PTEN, PCNA, c-myc, VEGF, MDR-1, Bcl-2, nm23, MMP-9, TIMP-1, CD44v6 proteins via immunocytochemistry staining, RRM1 and ERCC1 mRNA by real-time fluorescent quantitative-PCR. Results: The resis-tance index of H460/Gem' cells (the deputy of cells in the process of inducement) to gemcitabine was 1.201, and the cell line also exhibited cross-resistance to paclitaxol, fluorouraci, etoposide, cisplatin and oxaliplatin, but kept sensitivity to vinorelbine and taxotere. The doubling time of H460/Gem' cells was longer and figures in G0-G1 phase was decreased than that of NCI-H460 cells. Compared with NCI-H460 cells, H460/Gem' cells had achieved TIMP-1 protein expression emerged, nm23 protein expression enhanced, VEGF and MMP-9 protein expressions reduced, and CD44v6, P53 protein expressions van-ished, but expressions of EGFR, c-erb-B-2, PTEN, PCNA, c-myc, MDR-1, Bcl-2 proteins and RRM1, ERCC1 mRNA changed trivially. The resistance index of H460/Gem cells to gemcitabine was 1.644, and the cell line also exhibited cross-resistance to fluorouraci, cisplatin and oxaliplatin, but kept sensitivity to paclitaxol, vinorelbine, taxotere, and etoposide. The doubling time of H460/Gem cells was longer and figures in G0-G1 phase was decreased than those of NCI-H460 cells. The farther studies indicated that, compared with NCI-H460 cells, the expressions of MDR-1, nm23 and Bcl-2 proteins in H460/Gem cells had been enhanced, c-erb-B-2 protein expression emerged, P53, MMP-9 and VEGR protein expression had been weakened, but the changes of PTEN, PCNA, c-myc, TIMP-1, EGFR, CD44v6 protein, RRM1 mRNA and ERCC1 mRNA expressions were trivial. Furthermore, compared with its parental cells, H460/Gem cells were mixed with giant cells of different sizes that were larger and more irregular. Conclusion: The human gemcitabine-resistant non-small cell lung cancer cell line H460/Gem had achieved multi-drug resistance and great changes of biological characters compared with its parental cells. And these changes possibly participated in the formation of multidrug resistance.     

上海中医药大学附属普陀医院实验中心，上海中医药大学中西医结合肿瘤介入研究所，上海200062

背景与目的：细胞凋亡受阻是肿瘤细胞产生耐药的重要因素,从细胞凋亡的研究入手,将为肿瘤的治疗和耐药性的逆转开辟新的途径。本研究观察一种新型萘酰亚胺类衍生物8c对结肠癌HCT116/L-OHP耐药细胞的作用,探讨诱导HCT116/L-OHP耐药细胞凋亡的分子机制。方法：采用CCK-8法检测8c对HCT116/L-OHP细胞的增殖抑制作用;采用流式细胞术观察8c对HCT116/L-OHP细胞凋亡的影响;采用实时定量PCR(realtime PCR)检测p53 mRNA、Bax mRNA及Bcl-2 mRNA水平变化;蛋白［质］印迹法(Western blot)检测p-p53、Bax、Bcl-2及细胞色素c(Cyt-c)的蛋白表达水平。结果：8c抑制HCT116/L-OHP细胞增殖的半数抑制浓度(IC₅₀=8.16 μmol/L)低于阳性对照氨奈菲特(IC₅₀=28.37 μmol/L);药物作用后,8c诱导耐药细胞产生凋亡,凋亡通路中p53(Ser15)磷酸化水平上升,但p53 mRNA水平不受影响;Bax蛋白水平及mRNA水平明显上升,Bcl-2蛋白及mRNA水平下降,使得Bax/Bcl-2比例增加,同时8c又引起细胞色素c的释放。结论：8c通过磷酸化p53 Ser-15位点激活p53,随即激活细胞凋亡通路相关蛋白的表达,这可能是8c抑制结肠癌HCT116/L-OHP耐药细胞增殖的重要机制之一。8c具有良好的抗肿瘤及抗耐药潜力和开发应用前景。     

Expression of p53, Bcl-2 and Bax in cisplatin-induced apoptosis in testicular germ cell tumour cell lines.

We examined the sensitivity for cisplatin-induced apoptosis in a panel of four testicular germ cell tumour (TGCT) cell lines and monitored the cellular expression of the apoptosis-related proteins p53, Bcl-2 and Bax. Three of four TGCT cell lines (NT2, NCCIT and S2) were hypersensitive for cisplatin-induced apoptosis, while the TGCT cell line 2102 EP appeared to be resistant for cisplatin-induced apoptosis, even at relatively high drug concentrations (12.5 microM). For all four cell lines, the induction of apoptosis by cisplatin correlated with drug sensitivity in the MTT assay. The differences in chemosensitivity and induction of apoptosis could not be attributed to differences in cellular platinum accumulation, DNA platination or platinum-DNA adduct removal. We next analysed the relationship between p53 status and cisplatin-induced up-regulation of p53, and the susceptibility to cisplatin-induced apoptosis. Wild-type p53 containing NT2 and 2102 EP cells showed p53 up-regulation upon drug treatment, and NCCIT (mutant p53) and S2 (no p53 protein) cells did not. Consistently, the increase in wild-type p53 protein in NT2 and 2102 EP cells led to an increase in mRNA level of the p53 downstream gene p21/WAF/CIP, whereas mutant p53-containing NCCIT cells and p53-non-expressing S2 cells could not transactivate this p53-responsive gene. As NT2, NCCIT and S2 were readily triggered into apoptosis, while 2102 EP cells failed to undergo cisplatin-induced apoptosis, our data suggest that the presence of wild-type and/or transactivation-competent p53 might not be an absolute prerequisite for efficient induction of apoptosis in TGCT cell lines. Also endogenous levels of Bcl-2 and Bax expression did not correlate with cisplatin-induced apoptosis. In addition, the endogenous Bcl-2 and Bax expression was not affected by cisplatin treatment. The present study suggests that, at least in our panel of TGCT cell lines, hypersensitivity for cisplatin-induced apoptosis might not be necessarily correlated with the presence of wild-type p53 and is probably not associated with Bcl-2 and Bax expression.     

Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells

Cisplatin produces good responses in solid tumours including small cell lung cancer (SCLC) but this is limited by the development of resistance. Oxaliplatin is reported to show activity against some cisplatin-resistant cancers but there is little known about oxaliplatin in SCLC and there are no reports of oxaliplatin resistant SCLC cell lines. Studies of drug resistance mainly focus on the cellular resistance mechanisms rather than how the cells develop resistance. This study examines the development of cisplatin and oxaliplatin resistance in H69 human SCLC cells in response to repeated treatment with clinically relevant doses of cisplatin or oxaliplatin for either 4 days or 2 h. Treatments with 200 ng/ml cisplatin or 400 ng/ml oxaliplatin for 4 days produced sublines (H69CIS200 and H69OX400, respectively) that showed low level (approximately two-fold) resistance after eight treatments. Treatments with 1,000 ng/ml cisplatin or 2,000 ng/ml oxaliplatin for 2 h also produced sublines, however, these were not stably resistant suggesting shorter treatment pulses of drug may be more effective. Cells survived the first five treatments without any increase in resistance, by arresting their growth for a period and then regrowing. The period of growth arrest was reduced after the sixth treatment and the H69CIS200 and H69OX400 sublines showed a reduced growth arrest in response to cisplatin and oxaliplatin treatment suggesting that ‘regrowth resistance’ initially protected against drug treatment and this was further upregulated and became part of the resistance phenotype of these sublines. Oxaliplatin dose escalation produced more surviving sublines than cisplatin dose escalation but neither set of sublines were associated with increased resistance as determined by 5-day cytotoxicity assays, also suggesting the involvement of regrowth resistance. The resistant sublines showed no change in platinum accumulation or glutathione levels even though the H69OX400 subline was more sensitive to buthionine sulphoximine treatment. The H69CIS200 cells were cross-resistant to oxaliplatin demonstrating that oxaliplatin does not have activity against low level cisplatin resistance. Relative to the H69 cells, the H69CIS200 and H69OX400 sublines were more sensitive to paclitaxel and taxotere suggesting that the taxanes may be useful in the treatment of platinum-resistant SCLC. These novel cellular models of cisplatin and oxaliplatin resistant SCLC will be useful in developing strategies to treat platinum-resistant SCLC.     

Establishment and characterization of a paclitaxel-resistant human non-small cell lung cancer cell line

We have established a paclitaxel-resistant mutant cell line called H460/TAX which was derived from human non-small cell lung cancer (NSCLC) H460. A 64-fold greater resistant was shown in our assay as compared with the parental cells. High specificity of drug resistance was also observed since this mutant was not cross-resistant to several other anticancer drugs. Drug accumulation in H460/TAX was significantly less than that in H460. Many endogenous protein profiles were intact, including the expression level of P-glycoprotein, multidrug resistance-associated protein, the 70 kDa heat shock proteins as well as the phosphorylation of Bcl-2 in H460/TAX cells, except that the total amount of alpha- and beta- tubulins was higher in H460/TAX than in H460 cells. Higher drug concentration and longer treatment for paclitaxel were required in H460/TAX to exert the phosphorylation of keratin 19 which was then accompanied by reorganization of the intermediate filament and the microtubule networks. Since all of the aforementioned factors involved in paclitaxel-resistance in other systems were not found to be significantly altered in H460/TAX, there must be other paclitaxel-resistance mechanisms(s) which remains to be identified in human lung cancers.     

Analysis of mechanisms of Cisplatin resistance in 3 pairs of human tumor-cell lines expressing normal and resistant responses to Cisplatin

Three pairs of human tumour cell Lines each having a cisplatin sensitive parental cell line (normal) and a cisplatin resistant derivative were tested for their cisplatin responses and expression for three reputed mechanisms of resistance. In each case, the derivative cell lines showed resistance to cisplatin when treated either in exponential or plateau growth phase. For both the resistant and the normal cell line there was greater resistance to cisplatin treatment when cells were treated in exponential growth phase. Mechanisms of resistance were expressed in all three resistant variants. The cervical carcinoma resistant variant expressed higher GSH levels and lower cisplatin uptake levels but not elevated polymerase levels. The other two variant cell lines for the glioma and the lung cancer expressed all three of the mechanisms (increased GSH, decreased cisplatin uptake and increased polymerases) associated with resistance. There was no correlation to the level of resistance expressed and the number of mechanisms expressed but the change in response of the parental line to becoming a resistant variant may be related to the basal level of expression of some of the factors that are associated with resistance.     

吉西他滨耐药细胞系H460/Gem的建立及其生物学特性研究

目的:建立吉西他滨耐药细胞系H460/Gem,通过研究其生物学特性,初步从细胞水平分析其获得性耐药的可能机制。方法:以人肺大细胞癌细胞系NCI-H460为亲代细胞,采用"2/3临床血浆峰浓度间歇诱导"法,用吉西他滨进行诱导,建立耐吉西他滨的细胞亚系H460/Gem,把诱导过程中的细胞命名为H460/Gem。分别在诱导前、诱导过程中和诱导成功后的三个不同时间点,采用MTT法检测其耐药指数和对其他化疗药物的敏感性,使用倒置光学显微镜观察形态学特征,通过绘制生长曲线计算倍增时间,以流式细胞术分析细胞周期分布,以免疫细胞化学染色检测P53、EGFR、C-erbB-2、PTEN、PCNA、c-myc、VEGF、MDR-1、Bcl-2、nm23、MMP-9、TIMP-1和CD44v6蛋白的表达,采用实时荧光定量PCR法检测RRM1mRNA表达。结果:H460/Gem细胞对吉西他滨耐药指数为1.201时,对紫杉醇、氟尿嘧啶、依托泊甙、顺铂、奥沙利铂耐药,对长春瑞滨、多西他赛敏感;它的倍增时间(18.611小时)比NCI-H460细胞(16.731小时)稍延长,G0~G1期细胞减少(分别为51.56%和68.06%),VEGF、MMP-9表达减弱,失去CD44v6和P53表达,nm23表达增强,出现TIMP-1表达,EGFR、C-erbB-2、PTEN、PC-NA、c-myc、MDR-1、Bcl-2表达无变化,RRM1 mRNA的相对表达量有所减少(分别为0.1589和0.4510)。H460/Gem细胞对吉西他滨的耐药指数为1.644时,同时对氟尿嘧啶、顺铂和奥沙利铂耐药,而对紫杉醇、长春瑞滨、多西他赛和依托泊甙敏感,倍增时间(51.539小时)比NCI-H460细胞明显延长,G0~G1期细胞(57.23%)有所减少,MDR-1、nm23、Bcl-2表达增强,出现C-erbB-2表达,P53、MMP-9和VEGR表达减弱,PTEN、PCNA、c-myc、TIMP-1、EGFR和CD44v6表达无变化,RRM1 mRNA的相对表达量(0.3389)变化不大。与NCI-H460细胞相比,H460/Gem细胞逐渐呈现出如下形态学特征:形态不规则,大小不一,形态各异,且透光性减弱。结论:H460/Gem细胞的生物学特性较NCI-H460细胞发生了较大变化,其多药耐药性的发生与多种基因表达有关。     

CXCL4对非小细胞肺癌H460细胞顺铂耐药性的影响及其机制探讨

目的:探讨血小板因子4（CXCL4）对人非小细胞肺癌H460细胞顺铂耐药性的影响及其作用机制。方法:体外诱导法建立顺铂耐药的非小细胞肺癌细胞株H460/DDP,并鉴定其生物学特性。采用CCK-8法分别检测H460及H460/DDP对顺铂的耐药性。qRT-PCR及Western Blot检测亲本细胞株H460及顺铂耐药株H460/DDP中CXCL4的mRNA及蛋白表达水平。通过CCK-8法检测过表达或者敲低CXCL4后亲本细胞株H460或顺铂耐药株H460/DDP对顺铂的敏感性。最后利用Western Blot及qRT-PCR法探讨CXCL4抵抗非小细胞肺癌化疗敏感性的调控机制。结果:成功构建了非小细胞肺癌顺铂耐药株H460/DDP（H460/DDP IC50=55.005 μg/ml）,CCK-8结果提示顺铂耐药株H460/DDP与亲本细胞株H460细胞的增殖速度没有明显差异（P＞0.05）;顺铂耐药株H460/DDP中,CXCL4 mRNA及蛋白水平均显著增高（P＜0.000 1）;在亲本细胞株H460中稳定过表达CXCL4能显著降低其对顺铂的敏感性（P＜0.000 1）,而于顺铂耐药株H460/DDP中敲低CXCL4能显著增加H460/DDP对顺铂的敏感性（P＜0.000 1）;Western Blot及qRT-PCR结果显示,CXCL4能激活Wnt/β-catenin 信号通路,促进下游基因MYC、CyclinD1、MMP-7、CDK4的表达。结论:CXCL4通过调控Wnt/β-catenin 信号通路促进非小细胞肺癌对顺铂的耐药性。     

Increased accumulation of p53 protein in cisplatin-resistant ovarian cell lines

We have examined p53 protein levels in cell lines selected for resistance to the chemotherapeutic drug cis-diamminedichloroplatinum (II), cisplatin. The majority of the independent cisplatin-resistant clones isolated by a single selection with cisplatin from the ovarian tumour cell line A2780 showed increased levels of p53 protein compared to the parental cell line. Elevated p53 protein levels were also observed in cisplatin-resistant ovarian human tumour lines isolated after multiple exposures to cisplatin (A2780/cp70 and OVIP/DDP). Direct PCR sequencing of p53 cDNAs showed that both the A2780/cp70 and the parental A2780 cell lines had a wild-type p53 gene sequence. The OVI P and OVI P/DDP lines both had a heterozygous mutation at codon 126. Cell-cycle analysis after gamma-irradiation or cisplatin treatment showed evidence of a G1/S and G2/M cell-cycle checkpoint in both A2780/cp70 and the sensitive parental cell lines. However, the resistant cell line A2780/cp70 showed less inhibition of DNA synthesis after gamma-irradiation than the sensitive cell line. Transfection of a mutant p53 gene construct (containing a mutation at codon 143, val to ala) into the A2780/cp70 resistant cells conferred a significantly increased sensitivity to cisplatin, suggesting that p53 is a direct determinant of cisplatin resistance in these cells. However, expression of this mutant p53 in the A2780 cells did not affect sensitivity.     

Development of resistance to a trinuclear platinum complex in ovarian carcinoma cells

BBR3464 is a trinuclear platinum complex that exhibits a potent cytotoxicity and efficacy against cisplatin-resistant tumors. To better understand the determinants of cellular resistance to BBR3464, we selected a resistant ovarian carcinoma cell line after exposure to the complex. The resistant cells (A2780/BBR3464) exhibited a high level of resistance to the selecting agent, but a marginal cross-resistance to cisplatin. Although cellular accumulation of BBR3464 was similar in parental and in resistant cells, DNA platination was decreased in A2780/BBR3464 cells, suggesting a reduced drug accessibility to DNA. This behavior reflected a partial drug inactivation at cytoplasmic level, as a consequence of increased levels of nucleophilic molecules including metallothioneins and human neurofilament low, but not glutathione. A2780/BBR3464 cells also exhibited a reduced susceptibility to apoptosis, which was consistent with reduced expression of Bax, and an alteration of DNA mismatch repair system, as reflected by lack of expression of MLH1 and PMS2, which could impair the recognition/repair of DNA lesions. Whereas both platinum drugs induced G2/M arrest in the parental cells, BBR3464, but not cisplatin, caused a late G1 arrest of resistant cells. Cisplatin induced an appreciable increase of p21(WAF1) levels in both models, in contrast to BBR3464 that produced a substantial upregulation of p21(WAF1) only in parental cells. An inverse relationship with p21(WAF1) modulation was found for CHK1 in parental cells treated with both agents and in resistant cells treated with cisplatin. This pattern of response is consistent with a regulatory loop involving p53 and p21(WAF1) at G2 checkpoint. In contrast, no modulation of CHK1 was found in A2780/BBR3464 treated with the triplatinum compound. These findings, indicating a different activation of regulatory pathways at DNA damage checkpoints in response to cisplatin and BBR3464, support an altered ability of resistant cells to recognize or tolerate sublethal lesions induced by BBR3464.     

Phosphorylation of p53 on Thr55 by ERK2 is necessary for doxorubicin-induced p53 activation and cell death

We recently reported that exposure of human cervical carcinoma cells to doxorubicin results in extracellular signal-regulated kinase (ERK)2 activation, which in turn phosphorylates p53 on a previously uncharacterized site, Thr55. This study sought to clarify the biological significance of doxorubicin-induced Thr55 phosphorylation. In breast carcinoma MCF7 cells, doxorubicin (300 nM) activated ERK2 and induced phosphorylation of p53 on Thr55 residues. Pretreatment of MCF7 cells with an ERK2 chemical inhibitor, PD98059 or U0126, blocked doxorubicin-induced p53 activation and suppressed phosphorylation of p53Thr55. MCF55a cells were established by transfection of full-length p53 carrying Thr55 mutation (Thr to Ala) into MCF7 cells. Doxorubicin (500 nM) could not induce p53 activation in MCF55a cells, which showed significantly increased drug resistance toward doxorubicin. While the expression of the apoptotic protein, Bax, showed no difference between MCF7 and MCF55a cells, Bcl-2, an antiapoptotic protein, was constitutively expressed in MCF55a cells. The increase of Bcl-2 protein and/or Bcl-2/Bax ratio might at least partly contribute to the drug resistance of MCF55a cells. In summary, our results suggest that phosphorylation of p53Thr55 by ERK2 is important for doxorubicin-induced p53 activation and cell death.     

Synergistic Anticancer Activity of 5-Aminolevulinic Acid Photodynamic Therapy in Combination with Low-dose Cisplatin on Hela Cells

Objective: Photodynamic therapy (PDT ) is a promising modality for the treatment of various tumors.In order to assist in optimizing treatment, we applied 5-ALA/PDT in combination with low-dose cisplatin toevaluate cytotoxicity in Hela cells. Methods: Antiproliferative effects of 5-ALA/PDT and cisplatin, alone and incombination, were assessed using MTT assay. To examine levels of apoptosis, Hela cells treated with 5-ALA/PDT,and combination treatment were assessed with Annexin-V/PI by flow cytometry. To investigate the molecularmechanisms underlying alterations in cell proliferation and apoptosis, Western blot analysis was conducted todetermine the expression of p53, p21, Bax and Bcl-2 proteins. Results: MTT assays indicated that combinationtreatment obviously decreased the viability of Hela cells compared to individual drug treatment. In addition,it was confirmed that exposure of Hela cells to 5-ALA/PDT in combination with low-dose cisplatin resulted inmore apoptosis in vitro. Synergistic anticancer activity was related to upregulation p53 expression and alterationin expression of p21, Bcl-2 and Bax. Conclusion: Our findings suggest that administration of 5-ALA/PDT incombination with the low-dose cisplatin may be an effective and feasible therapy for cervical cancer.     

Upregulation of Bcl-2 is associated with cisplatin-resistance via inhibition of Bax translocation in human bladder cancer cells

The efficacy of cisplatin in cancer chemotherapy is limited by the development of resistance. To elucidate the molecular basis of resistance to cisplatin, we compared cisplatin-induced apoptotic responses of the parental human bladder cancer cell line, T24 and its resistant subclone, T24R2. In T24 cells, cisplatin induce apoptosis and the activation of caspase-8, -9 and -3 and poly(ADP-ribose) polymerase cleavage. The expression levels of Fas, FasL, and FADD were not changed by the treatment with cisplatin. Furthermore, neither Fas-neutralizing antibody nor dominant negative mutant of FADD affected cisplatin-induced apoptosis. Western blot analysis of subcellular fractions showed that cisplatin induced redistribution of Bax and cytochrome c. Thus, cisplatin causes apoptosis in a death receptor-independent and mitochondria-dependent fashion in T24 cells. In contrast, overexpressed Bcl-2 protein inhibited cisplatin-induced Bax translocation and its downstream events in T24R2. Downregulation of Bcl-2 by RNAi potentiated the redistribution of Bax and cytochrome c and reversed cisplatin-resistance. Our results indicate that upregulation of Bcl-2 contributes to the development of cisplatin-resistance and usage of siRNA which targets the Bcl-2 gene may offer a potential tool to reverse the resistance to cisplatin in bladder cancer.     

The novel trinuclear platinum complex BBR3464 induces a cellular response different from cisplatin

BBR3464 is a new platinum-based drug non cross-resistant with cisplatin. To characterise the cellular basis of BBR3464 cytotoxicity as opposed to cisplatin, we performed a comparative study of the two drugs in cisplatin-resistant neuroblastoma and astrocytoma cells. In both model systems, BBR3464 proved to be more potent than cisplatin and was able to overcome cisplatin resistance. The higher potency exhibited by BBR3464 correlated with an increased cellular platinum accumulation and DNA-adduct formation. At equitoxic doses, BBR3464 induced apoptosis to a lesser extent than cisplatin and failed to overcome the decreased susceptibility to cisplatin-induced apoptosis in cisplatin-resistant cells. Cell cycle analysis showed a dose-dependent G2/M arrest by BBR3464. In astrocytoma cells, cisplatin treatment resulted in the upregulation of p53, p21 and bax, while only p21 induction was observed after BBR3464 treatment. In cisplatin-resistant cells, the reduced sensitivity to cisplatin paralleled a resistance to the induction of p53/p21 pathway by cisplatin, while the same doses of BBR3464 induced p21 to a similar extent in the resistant cells as in the parental cells. In conclusion, BBR3464 induces a cellular response that is different from cisplatin, supporting the view that the two drugs act through different mechanisms. Our data indicate that BBR3464 may be a promising agent in the treatment of tumours unresponsive to cisplatin and with a non-functional p53.     

Reversion of multidrug resistance by SKI-II in SGC7901/DDP cells and exploration of underlying mechanisms

In order to investigate whether SKI-II could reverse drug resistance and its possible mechanisms, we treated SGC7901/DDP cells with SKI-II or SKI-II in combination with DDP. Then cell growth, apoptosis, micro- morphological changes, and expression of SphK1, P-gp, NF-kB, Bcl-2 and Bax were assessed by MTT assay, flow cytometry, electron microscopy, immunocytochemistry and Western blot assay respectively. SGC7901/DDP cells were insensitive to cisplatin 2.5 mg/L, but when pretreated with SKI-II, their proliferation was inhibited by cisplatin 2.5mg/L significantly, the inhibition rate increasing with time and dose. The apoptosis rate was also significantly elevated. Expression of SphK1 and P-gp was decreased significantly, Pearson correlation analysis showing significant correlation between the two (r=0.595, P<0.01). Expression of NF-kB and Bcl-2 was decreased significantly, while that of Bax was increased, compared to the control group. There were significant correlations between SphK1 and NF-kB(r=0.723, P<0.01), and NF-kB and Bcl-2(r=0.768, P<0.01). All these data indicated that SKI-II could reverse drug resistance of SGC7901/DDP to cisplatin by down-regulating expression of P-gp and up-regulating apoptosis through down-regulation of SphK1. The increased apoptotic sensitivity of SGC7901/ DDP to cisplatin was due to the decreasing proportion of Bcl-2/Bax via down-regulating NF-kB.     

Gankyrin基因沉默对人卵巢癌SKOV3/CDDP细胞顺铂耐药性的逆转作用和机制

背景与目的：卵巢癌是常见的妇科肿瘤,抗肿瘤药耐药性的产生是卵巢癌治疗失败的主要原因之一,Gankyrin基因被认为与肿瘤耐药性密切相关,本文探讨了Gankyrin基因沉默对卵巢癌耐顺铂细胞系SKOV3/DDP顺铂耐药性的逆转作用及机制。方法：应用real-time PCR技术考察Gankyrin在SKOV3和SKOV3/DDP细胞中的表达,应用MTS法检测Gankyrin对SKOV3/DDP细胞顺铂耐受性的影响,应用流式细胞术检测肿瘤细胞凋亡和细胞内罗丹明-123(Rhodamine-123,Rh-123)含量的变化,Western blot和real-time PCR技术检测肿瘤细胞耐药相关蛋白MDR1、Caspase-3/8、Survivin和Bcl-2蛋白表达,Western blot法检测p53、NF-κB和PTEN蛋白表达和AKT磷酸化水平。结果：Gankyrin在SKOV3/DDP细胞中表达升高,沉默Gankyrin基因后可增加SKOV3/DDP细胞对顺铂的敏感性。基因沉默前后耐药逆转倍数(resistant factor,RF)为1.81和2.45,肿瘤细胞中Rh-123含量提高了1.73和2.42倍,细胞凋亡率是对照组的2.23倍和4.23倍,耐药相关蛋白MDR1、Survivin和Bcl-2蛋白水平显著下降,MDR1 mRNA表达是对照组的62.8%和21.6%,Survivin mRNA表达是对照组的24.5%和10.3%,Bcl-2 mRNA表达是对照组的47.5%和18.4%,Caspase-3/8、p53和PTEN表达水平上升,AKT磷酸化和NF-κB水平下降。结论：沉默Gankyrin基因可逆转SKOV3/DDP对顺铂的耐药性,可能与抑制药物外排,促进细胞凋亡有关,PTEN/AKT/NF-κB/p53信号通路可能是其中心环节。     

人大细胞肺癌多药耐药株H460/cDDP的建立及其生物学特性

目的：建立人大细胞肺癌多药耐药株H460/cDDP．并对其生物学特性进行测定。方法：以人大细胞肺癌细胞株H460为亲本细胞．采用顺铂大剂量问歇诱导法建立多药耐药细胞株H460／cDDP。光镜下观察其形态学变化；MTT法测定药物敏感性；锥虫蓝拒染实验测定细胞生长曲线、倍增时间；进行染色体核型分析；棵鼠成癌实验测定其体内成瘤活性。结果：历时近6个月建成人大细胞肺癌多药耐药株H460／cDDP．经鉴定其对顺铂的耐药指数为10．21，对5-氟尿嘧啶，多柔比星(阿霉素)、依托泊苷(足叶乙甙)、甲氨蝶呤有不同程度的交叉耐药。H460／cDDP较亲本细胞分裂高峰延迟，倍增时间由20．78小时延长至3646小时．细胞形态改变，染色体变化不突出。经过反复冻存．复苏．上述特性保持稳定．并具有体内成瘤活性，成瘤时间较亲本细胞有所延迟。结论：新建大细胞肺癌多药耐药株H460／cDDP呈中等度耐药、多药耐药表型稳定且具有体内成瘤活性，可用于下游实验。     

Mechanisms of apoptosis induced by the synthetic retinoid CD437 in human non-small cell lung carcinoma cells.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been shown to induce apoptosis in various tumor cell lines including human non-small cell lung carcinoma (NSCLC) cells, which are resistant to the natural all-trans retinoic acid and to many synthetic receptor-selective retinoids. Although the mechanism of this effect was not elucidated, it was found to be independent of nuclear retinoid receptors. In the present study, we analysed the mechanisms by which CD437 induces apoptosis in two human NSCLC cell lines: H460 with wild-type p53 and H1792 with mutant p53. Both cell lines underwent apoptosis after exposure to CD437, although the cell line with wild-type p53 (H460) was more sensitive to the induction of apoptosis. CD437 increased the activity of caspase in both cell lines, however, the effect was much more pronounced in the H460 cells. The caspase inhibitors (Z-DEVD-FMK and Z-VAD-FMK) suppressed CD437-induced CPP32-like caspase activation and apoptosis in both cell lines. CD437 induced the expression of the p53 gene and its target genes, p21, Bax, and Killer/DR5, only in the H460 cells. These results suggest that CD437-induced apoptosis is more extensive in NSCLC cells that express wild-type p53, possibly due to the involvement of the p53 regulated genes Killer/DR5, and Bax although CD437 can also induce apoptosis by means of a p53-independent mechanism. Both pathways of CD437-induced apoptosis appear to involve activation of CPP32-like caspase.